Cloning of Celluase Gene cel6B from Lentinula Endodes and Its Expression in Escherichia Coil
|Keywords||Lentinula edodes Cellulase cel6B gene Congo red RT-PCR|
Cellulose is a kind of biomass energy produced by photosynthesis of plant. Theoretically, one ton of crop straws can manufacture 1166.7 tons of ethanol. Besides, cellulose could be substituted for petroleum producing gasoline and diesel, which is important for development and utilization of renewable energy resources. By estimate, there is 1 700 tons of biomass produced by photosynthesis every year, which is almost 10 times of global energy consumption per year. However, less than 1% of the biomass is utilized as energy resources. It is the water-insoluble high crystalline structure and the lignose wraps it that hinder degradation of cellulose. And the low degradation leads to low utilization of cellulose. Hydrolysis Method utilizes cellulase to decompose cellulose into glucose, which can be recycle resource, relieve pollution and achieve sustainable use of resources. Researches on mechanism of degradation of cellulose have been carried out a lot, but there are still many unclear details in the process of cellulose being decomposed and conversed into glucose by cellulase. In this study, Lentinula edodes cultivated in liquid PDA culture medium was used to extract total RNA. Cellulase gene cel6B was cloned by RT-PCR, and prokaryotic expression vector pET28a-cel6B was successfully constructed. The vector pET28a-cel6B was transducted into E.coli BL21(DE3) to construct engineering bacteria with IPTG inducing protein expression. Results of SDS-PAGE shows that cel6B gene encoded 46.4 kDa protein CEL6B with the induction of IPTG. The engineering bacteria were inoculated into liquid congo-red culture medium with CMC-Na as unique carbon source and cultured.7 days later, color of congo-red turned light, which preliminarily proved that cellulase CEL6B possesses the ability to decompose cellulose.